The present invention relates to a class of perfluorinated esters of alkanoyl L-carnitine and their use as cationic lipids suitable for favouring the intracellular delivery of pharmacologically active compounds, facilitating their transmembrane transport, or for promoting their interaction with specific cell membrane sites (receptors).
What is meant by xe2x80x9cintracellular deliveryxe2x80x9d is cellular transfection with polynucleotides endowed with therapeutic action and the introduction of antiviral drugs or immunogenic polypeptides into the cells.
Many of the pharmacologically active substances, such as, for instance, polypeptides and proteins or drugs in general need to penetrate into the cells to exert their effects by influencing cell functions at subcellular or molecular level. For these molecules the cell membrane constitutes a selectively impermeable barrier. The cell membrane, in fact, performs a protective function, preventing the entry of potentially toxic substances, but also that of compounds with therapeutic activity. The complex composition of the cell membrane includes phospholipids, glycolipids and proteins; its function is influenced by cytoplasmatic components such as Ca++ and other ions, ATP, microfilaments, microtubules, enzymes and proteins that bind Ca++. The interaction between the structural and cytoplasmatic components of the cells and the response to external signals are responsible for the selectivity shown by and the various different cell types. The barrier effect of the membranes can be overcome by combining substances in complexes with lipid formulations that reproduce the composition of naturally occurring membrane lipids. These lipids are capable of fusing with the membranes and of releasing the substances combined with them into the cells. The lipid complexes are capable not only of facilitating intracellular transfer by means of fusion with the membranes, but can also diminish the charge repulsion between the membrane and the molecule that has to penetrate into the cell. Amphipathic lipids, such as membrane phospholipids, form lipid vesicles or liposomes in the aqueous systems.
Liposomes are vesicles in which an aqueous volume is entirely enclosed by one or more membranes composed of lipid molecules, usually phospholipids. Phospholipids, which consist in a hydrophilic head and a pair of carbon chains (hydrophobic tail), are the main components of biological membranes. In aqueous solution the hydrophobic tails autoassociate to exclude water, while the hydrophilic heads interact with the medium, spontaneously forming populations of vesicles of varying diameters. The lipids are generally zwitterionic, neutral or anionic. These vesicles can be used as carriers of drugs, small molecules, proteins, nucleotides and plasmids.
Over recent years, the cationic liposomes, a class of positively charged vesicles prepared from synthetic lipids, have been extensively used for the transfer of genetic material into the cells. The negative charge of DNA can interact with the positive charges of the cationic lipids, forming a stable DNA-liposome complex. The simplicity and versatility of this technology have made liposomes an important vehicle for the delivery of genes for gene therapy in human subjects. Currently, most of the vectors used for gene therapy and approved by the NIH Recombinant Advisory Committee include viral and synthetic systems.
Viral infection involves a series of complex mechanism in order to be able to attack a specific cell and carry the DNA into the nucleus. The rationale for the use of viral vectors for gene therapy is based on the possibility of replacing the viral genes with genes that code for a therapeutic function, while eliminating the ability of the viral particle to infect the cells. The limitations of viral therapy have to do with those viral elements that may be immunogenic, cytopathic and recombinogenic.
Great hopes are placed in the use of cationic lipids for gene therapy. These vectors possess great potential compared with those of biological origin, since they are much safer, less toxic and are also capable of incorporating genes of large size. As compared with biological-type vectors, however, they have a low intracellular gene transcription yield. It should be borne in mind, however, that the use of such transfection system is in an initial stage of research. Cationic lipids play a very important role in the formation of the DNA-lipid complex, in cell-complex interaction, in fusion with the membrane, in DNA release inside the cell and in transcription.
There are important examples of in-vivo applications of cationic liposomes. The first clinical trial on gene therapy was conducted by introducing an expression vector containing the human liposome-complexed HLA-B7 gene for the treatment of melanoma. Another important application relates to the treatment of pulmonary cystic fibrosis by means of the administration via the pulmonary route or as a nasal spray of the liposome-complexed expression vector SV-40C-FTR. Other clinical trials involving the use of liposomes in gene therapy for cancer are currently in progress.
Four constituent elements are generally identified in the structure of cationic lipids: the positively charged cationic head, the spacer, the anchor lipid and the linker bond.
The cationic head is responsible for the interactions between cationic liposomes and DNA, between the DNA-liposome complex and the cell membrane and the other components of the cell. It consists of mono- or polycationic groups (depending on the number of charges) that can be variably substituted.
The spacer is part of the molecule that separates the cationic head from the hydrophobic tail and is involved in ensuring optimal contact between the cationic head and the negative charges of the DNA phosphates.
The anchor lipid is the non-polar hydrocarbon part of the molecule and determines the physical properties of the double lipid layer, such as its rigidity and rate of exchange with membrane lipids.
What is meant by xe2x80x9clinker bondxe2x80x9d is the bond between the hydrocarbon chains and the rest of the molecule. This bond determines the chemical stability and biodegradability of the cationic lipids.
The scientific and patent literature is rich in references to the preparation and use of liposomes: however, only patent application EP 0 279 887 A2 describes the use of a derivative of carnitine, i.e. phosphatidyl carnitine, optionally in mixtures with other phospholipids and lipids (cholesterol, phosphatidyl choline, phosphatidyl serine), for the preparation of liposomes.
In the only example provided regarding the preparation of liposomes, liposomes of phosphatidyl carnitine are produced which incorporate propranolol, and drug known to be active as an antihypertensive, anti-angina and antiarrhythmia agent. The carnitine derivative is used here on account of the pronounced myocardial tropism of carnitine. This tropism makes it possible to avoid the liposomes being metabolised by the liver, rather than reaching the desired target site.
The presence of phosphatidyl carnitine also makes it possible to administer the liposomes orally, since they are resistant to intestinal lipases.
It has now been found that cationic lipids with a potent action favouring intracellular delivery of biologically active compounds consist in the perfluorinated ester of alkanoyl L-carnitine with the following formula (I): 
where:
R1 is alkanoyl, linear or branched, with 2-20, and preferably 4-12 carbon atoms, optionally perfluorinated
R2 is perfluorinated alkyl, linear or branched, with 4-20, and preferably 5-12, carbon atoms; and
X is the anion of a pharmacologically acceptable acid.
Therefore, it is an object of the present invention cationic liposomes consisting of perfluorinated esters of L-carnitine of formula (I). Esters of formula (I) are new, accordingly, they represent a further object of the present invention, together with their use in the preparation of cationic liposomes.
Another object of the present invention is the use of a cationic liposome as above defined for the preparation of a medicament useful for the intracellular delivery of a pharmacologically active compound, said medicament being also useful for promoting the interaction of a pharmacologically active compound with cell membrane receptors. In particular, according to the present invention, the pharmacologically active compound is a gene, optionally comprised in a suitable vector. Therefore, the medicament provided by the present invention is useful for gene therapy, for example wherein said gene is xcex2-gal.
Examples of R1, though not exclusively these are: acetyl, propionyl, butyryl, valeryl, isovaleryl, undecanoyl, lauroyl, tridecafluoroheptanoyl, heptadecafluorononanoyl, heptacosafluoromyristoyl, pentadecafluoro-octanoyl and 5H -octafluoropentanoyl.
What is meant here by xe2x80x9cperfluorinatedxe2x80x9d R2 is an alkyl in which at least 40% of the hydrogen atoms are replaced by fluorine atoms. Examples of such alkyls, though not exclusively these, are:
1,1H-2,2H-tridecafluoro-octyl;
1,1H-2,2H-3,3H-pentafluoropentyl;
1,1H-2,2H-nonafluorohexyl;
1,1H-2,2H-3,3H-4,4H-5,5H-6,6H- nonafluorodecyl;
1,1H-2,2H-heptadecafluorodecyl;
1,1H-2,2H-heinicosafluorododecyl; and
1,1H-tricosafluorododecyl.
What is meant by pharmacologically acceptable acid is the anion of an acid that does not give rise to unwanted toxic or side effects.
These acids are well known to pharmacologists and experts in pharmaceutical technology.
Examples of these anions, though not exclusively the ones listed, are: chloride: bromide; iodide; aspartate; acid aspartate; citrate; acid citrate; tartrate; mucate; phosphate; acid phosphate; fumarate; acid fumarate; glycerophosphate; glucose phosphate; lactate; maleate; acid maleate; orotate; oxalate; acid oxalate; sulphate; acid sulphate; trichloroacetate; trifluoroacetate and methane sulphonate.
Here below are provided a number of non-exclusive examples of the preparation of compounds according to the invention described herein.